Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature.

Ling Y, Hayano S, Novick P - Mol. Biol. Cell (2014)

Bottom Line:
Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane.We show here that in yeast the oxysterol-binding proteins Osh1-Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function.This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p.

Figure 3: The polarized localization of Osh4 is dependent on PI4P. (A) Localization of Osh4-3xGFP in stt4ts cells and pik1ts cells at indicated temperatures. Osh4-3xGFP is mislocalized in pik1ts cells at the nonpermissive temperature. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. Scale bar, 5 μm. (B) Quantification of stt4ts cells and pik1ts cells with regard to polarized localization of Osh4-3xGFP at 25 or 37°C for 1 h. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments. Note that there were no pik1ts cells observed with polarized Osh4-3xGFP at 37°C. (C) Localization of Osh4-3xGFP point mutants defective in PI4P binding in cells. Scale bar, 2 μm. (D) Quantification of cells with polarized localized Osh4-3xGFP point mutant proteins. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments. Note that there were no large-budded cells observed with polarized Osh4 K396A-3xGFP or Osh4 R344A-3xGFP. (E) Steady-state expression levels of Osh4-3xGFP, Osh4K336A-3xGFP, and Osh4R344A-3xGFP. Yeast whole-cell lysates were prepared from strains expressing different Osh4 mutant proteins as described in Materials and Methods. Adh1p was followed as a loading control. (F) Complementation assays of Osh4p point mutants. The osh1-7Δ /CEN osh4ts cells were transformed with plasmids expressing various mutant Osh4p proteins as indicated. Serial dilutions of yeast cells were grown on –Ura plates to retain plasmids at 25 or 37°C for 3 d; only cells harboring functional OSH4 plasmids grew on plates.

Mentions:
Osh4p binds to both ergosterol and PI4P (Im et al., 2005; de Saint-Jean et al., 2011). A prior study demonstrated that ergosterol is important for Osh4p localization (Alfaro et al., 2011). We speculated that the polarized localization of Osh4p might also be dependent on binding to PI4P, based on PI4P's cellular distribution. To test this hypothesis, we first determined whether the localization of Osh4 is affected by the loss of enzymatic activities that synthesize PI4P. In budding yeast, there are two major PI 4-kinases responsible for PI4P synthesis. Pik1 is a Golgi-localized lipid kinase, whereas Stt4 functions at the plasma membrane (Audhya et al., 2000). Each of these two PI 4-kinases synthesizes ∼50% of the total PI4P in cells. We examined the localization of Osh4-3xGFP in pik1ts and stt4ts cells, respectively. At the permissive temperature, Osh4-3xGFP localized normally in both pik1ts cells and stt4ts cells. Of interest, upon shifting to the nonpermissive temperature for 1 h, Osh4-3xGFP appeared predominantly cytoplasmic in pik1ts cells, whereas its localization was normal in stt4ts cells (Figure 3, A and B). Subcellular fractionation results further support the proposal that Pik1p function is important for Osh4p membrane association (Supplemental Figure 2, A and B). Together these results suggest that the localization of Osh4p is controlled by the Pik1-generated pool of PI4P.

Figure 3: The polarized localization of Osh4 is dependent on PI4P. (A) Localization of Osh4-3xGFP in stt4ts cells and pik1ts cells at indicated temperatures. Osh4-3xGFP is mislocalized in pik1ts cells at the nonpermissive temperature. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. Scale bar, 5 μm. (B) Quantification of stt4ts cells and pik1ts cells with regard to polarized localization of Osh4-3xGFP at 25 or 37°C for 1 h. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments. Note that there were no pik1ts cells observed with polarized Osh4-3xGFP at 37°C. (C) Localization of Osh4-3xGFP point mutants defective in PI4P binding in cells. Scale bar, 2 μm. (D) Quantification of cells with polarized localized Osh4-3xGFP point mutant proteins. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments. Note that there were no large-budded cells observed with polarized Osh4 K396A-3xGFP or Osh4 R344A-3xGFP. (E) Steady-state expression levels of Osh4-3xGFP, Osh4K336A-3xGFP, and Osh4R344A-3xGFP. Yeast whole-cell lysates were prepared from strains expressing different Osh4 mutant proteins as described in Materials and Methods. Adh1p was followed as a loading control. (F) Complementation assays of Osh4p point mutants. The osh1-7Δ /CEN osh4ts cells were transformed with plasmids expressing various mutant Osh4p proteins as indicated. Serial dilutions of yeast cells were grown on –Ura plates to retain plasmids at 25 or 37°C for 3 d; only cells harboring functional OSH4 plasmids grew on plates.

Mentions:
Osh4p binds to both ergosterol and PI4P (Im et al., 2005; de Saint-Jean et al., 2011). A prior study demonstrated that ergosterol is important for Osh4p localization (Alfaro et al., 2011). We speculated that the polarized localization of Osh4p might also be dependent on binding to PI4P, based on PI4P's cellular distribution. To test this hypothesis, we first determined whether the localization of Osh4 is affected by the loss of enzymatic activities that synthesize PI4P. In budding yeast, there are two major PI 4-kinases responsible for PI4P synthesis. Pik1 is a Golgi-localized lipid kinase, whereas Stt4 functions at the plasma membrane (Audhya et al., 2000). Each of these two PI 4-kinases synthesizes ∼50% of the total PI4P in cells. We examined the localization of Osh4-3xGFP in pik1ts and stt4ts cells, respectively. At the permissive temperature, Osh4-3xGFP localized normally in both pik1ts cells and stt4ts cells. Of interest, upon shifting to the nonpermissive temperature for 1 h, Osh4-3xGFP appeared predominantly cytoplasmic in pik1ts cells, whereas its localization was normal in stt4ts cells (Figure 3, A and B). Subcellular fractionation results further support the proposal that Pik1p function is important for Osh4p membrane association (Supplemental Figure 2, A and B). Together these results suggest that the localization of Osh4p is controlled by the Pik1-generated pool of PI4P.

Bottom Line:
Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane.We show here that in yeast the oxysterol-binding proteins Osh1-Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function.This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p.